Studies in this project indicate powerful chemical reactivities expressed by antibodies (Abs), which in some instances results in catalytic hydrolysis of peptide bonds by a covalent mechanism reminiscent of conventional serine proteases. The long-term goals are to: (a) define immunological pathways underlying development of Ab chemical reactivity, (b) delineate the functional role of catalytic Abs in autoimmune disease using covalently reactive antigen analogs (CRAs) that can potentially serve as inhibitors of pathogenic Abs; and, (c) induce the synthesis of antigen-specific proteolytic antibodies by immunization with CRAs. In Aim 1, haptenic CRAs reactive with innate Ab active sites and peptidyl CRAs reactive with adaptively matured Ab active sites will be prepared. The peptidyl CRAs are derived from the sequence of the immunoregulatory neuropeptide VIP, catalytic Abs to which are found in autoimmune disease and are available for further study in polyclonal and recombinant forms. Aim 2 will study the covalent binding of haptenic CRAs and VIP-CRAs by surface Ig on splenocytes from immunologically naive mice and mice immunized with VIP-CRA and underivatized VIP. This will help identify nucleophilic B lymphocyte subpopulations in the innate repertoire and adaptive maturational pathways, confirm the molecular identity of the nucleophilic Ig and its cellular localization, and determine the sequence characteristics of Ig V genes expressed by nucleophilic B cells. Aim 3 will study induction of VIP-specific proteolytic Abs by immunization with VIP-CRA and control VIP. The hydrolysis of VIP and irrelevant peptides by monoclonal Abs from the animals will be analyzed, including Abs derived from splenocyte subpopulations expressing high level VIP-CRA binding. In Aim 4, the identity Ab nucleophilic residues at which covalent binding occurs will be determined, the possibility of conserved sequences and conserved 3-dimensional motifs associated with the nucleophiles will be explored, and structural constituents of the Ab active site responsible for the interplay of traditional noncovalent binding and chemical reactivity will be studied. Successful execution of this project will provide insight to the genesis of chemically reactive Abs and potentially validate the use CRAs for inhibition and induction of antigen-specific catalytic Abs. [unreadable] [unreadable] [unreadable] [unreadable]